Download Effects of maternal serum IgG anti-A (B) and

Biomedical Research 2017; 28 (4): 1855-1858
ISSN 0970-938X
www.biomedres.info
Effects of maternal serum IgG anti-A (B) and neonatal direct anti globulin
red blood cell antibody expression on the occurrence and development of
neonatal ABO haemolysis.
Ming Chen*, Yu Zeng, Xiaohe Chen
Department of Blood Transfusion, the People's Hospital of Ruian, Ruian, China
Abstract
Objectives: To explore the expression of maternal serum IgG anti-A (B) and neonatal direct anti
globulin, free antibody and Red Blood Cell (RBC) antibody on the occurrence and development of ABO
Haemolytic Disease of the New-born (ABO-HDN) and provide a valuable reference for the early
diagnosis of the disease.
Methods: we selected 546 cases of jaundice and hyperbilirubinemia neonates in our hospital from
February 2013 to February 2016, tested the maternal serum IgG anti-A (B) titer, neonatal direct anti
globulin, free antibody and RBC antibody, calculated the prevalence ratio of ABO haemolytic disease
and analysed the effects of these indexes in the occurrence and development of ABO-HDN.
Results: Among 546 cases, 227 cases were diagnosed as ABO haemolytic disease, the prevalence ratio
being 41.6%. The prevalence rate of ABO haemolytic disease of group A and group B were not
statistically significant (P>0.05). Pearson correlation analysis showed that there was a positive
correlation of pregnant women's serum IgG anti-A (B) titer and neonatal ABO haemolytic rate (r=0.883,
P<0.01). The serum IgG anti-A (B) titer level of pregnant women was not significantly correlated with
the severity of the disease. The difference was not statistically significant (P>0.05). Among all the 227
patients with ABO haemolytic disease, the positive rate of RBC antibody testing was 100.0%, which is
higher than that of the free antibody testing 74.9%. The difference was statistically significant (P<0.05)
and the positive rate of free antibody testing was also higher than that of direct anti globulin testing
14.9%. The difference was statistically significant (P<0.05).
Conclusion: The combined detection of pregnant women's serum IgG anti-A (B) and neonatal
erythrocyte antibody expression is expected to provide evidence for the clinical early diagnosis of ABOHDN. At the same time, with the increase of the titer of pregnant women's serum IgG anti-A (B), risks
of ABO-HDN will gradually increase, which should be paid more attention.
Keywords: IgG anti-A (B), Direct anti globulin, Free antibody, Red blood cell antibody, ABO haemolytic disease of the
new born.
Accepted on September 30, 2016
Introduction
The ABO haemolytic disease is a common Haemolytic Disease
of the New-born (HDN), which is caused by the blood group
incompatibility of mother and foetus. The clinical
manifestations usually include jaundice, haemolytic, anaemia,
bilirubin encephalopathy, some even have occurrence of
oedema, heaptosplenomegaly, ascites and pleural effusion with
a high rates of disability and death [1]. Currently, it still lacks
definite standards for the prenatal diagnosis and intervention of
ABO-HDN. Some scholars believed that it had some effects on
the prenatal diagnosis of HDN to test the maternal serum IgG
anti-A (B) titer, but other researchers pointed that IgG antibody
titer change only had limited directive effects on the clinical
preventive treatment [2]. We tried to combine neonatal direct
anti globulin, free antibody and RBC antibody to the laboratory
Biomed Res- India 2017 Volume 28 Issue 4
testing of ABO-HDN on the basis of the maternal serum IgG
anti-A (B) titer testing and some references [3], and achieved
satisfactory results.
Materials and Methods
Criteria for selection and exclusion
All the samples were selected from the ABO haemolytic
disease neonates in our hospital from February 2013 to
February 2016. A sample of infants who have symptoms of
jaundice and hyperbilirubinemia and who were single foetuses
with no more than 7 days after born and whose maternal
dipotassium ethylene diaminetetra-acetate (K2EDTA) blood
samples were well preserved and whose mothers were of blood
group O and their spouses non-O blood type were selected for
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Chen/Zeng/Chen
enrolment in the study. Neonates who were born ≤ 37 weeks
gestation to mothers or their blood specimens were ≥ 48 h for
testing or their mother was irregular antibody-positive or had
transfusion records or liver and kidney dysfunction, blood or
immune system diseases were excluded. At last, 546 babies
met the criteria were enrolled in the study.
Testing
Diagnosis of ABO haemolytic disease: ABO haemolytic
disease was diagnosed as described in literatures such as:
hyperbilirubinemia
[4],
maternal-foetal
ABO/Rh
incompatibility with direct anti globulin test positive or
antibody released test positive.
Maternal serum IgG anti-A (B) titer: We treated 200 μl
maternal serum by adding 200 μl 2-mercaptoethanol and put
the mixture at 37˚C water bath for 30 min. The serum after
treatment were added to each tube by the double-dilution
method with a titer of 1:16, 1:32, 1:64, 1:128, 1:512, 1:1024,
1:2048 respectively. The samples within a titer of 1:64 and
1:2048 were placed in labelled anti-human globulin cards,
added 1% erythrocyte suspension of the pregnant women’s
spouses or of the same blood group to them, then incubated for
15min at 37˚C environment, centrifuged 5 min for observation.
The results are positive when the red cell clots were located
within the gel or on the gel surface and negative when they
were at the bottom of the gel; the reference value for positive is
≥ 1:64 [5].
Direct anti globulin: We took 3 ml neonatal anticoagulant,
separated the plasma, diluted with saline and formulated 1%
erythrocyte suspension. 50 μl suspension was placed in the
HDN test card, centrifuged 2 min at 900 r/min and then 3 min
1500 r/min. When there was visible agglutination, the result
was positive and negative on the contrary [6,7].
Free antibody: 300 μl neonatal anticoagulant was taken and
put in three tubes by adding group-A, group-B, group-O
reagents respectively and 50 μl RBC in each tube, then
incubated for 30 min at 37˚C environment washed with saline
and got the supernatant. 100 μl anti-human globulin reagent
was added to each tube and centrifuged the mixture at 3400
r/min for 15 sec, the result was observed by the criteria for
direct anti globulin.
RBC antibody: We prepared 150 μl neonatal serum and 150 μl
RBC dispersion solution, placed them in 3 tubes respectively,
1% group-A erythrocyte suspension was added to No. 1 and
No.4 tubes, while 1% group-B erythrocyte suspension was
added to No. 2 and No. 5 tubes and 1% group-O erythrocyte
suspension was added to No. 3 and No. 6 tubes, and put the
mixture at 37˚C water bath for 15 min, centrifuged and
observed the result with the same criteria as direct anti
globulin.
Statistical analysis
Statistical analysis was conducted by SPSS18.0. For count
data, we used n% to express and category variables were
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compared using χ2 analysis. Measurement data was expressed
by x ± s. Continuous variables with a normal distribution were
compared by t-test, in others M(Q1, Q3) were used and
compared using Wilconx. Significance was defined as a p
value<0.05.
Results
Diagnosis
Among 546 cases, 227 cases were diagnosed as ABO
haemolytic disease, the prevalence ratio was 41.6%. The
prevalence ratio of ABO haemolytic disease of A blood type
and B blood type were not statistically significant (P>0.05,
Table 1).
Table 1. Prevalence ratio of ABO haemolytic disease of blood group A
and B.
Blood group ratio Cases
(%)
ABO haemolytic Prevalence
disease (n)
Group A
231
92
39.8
Group B
315
135
42.9
Total
546
227
41.6
Maternal serum IgG anti-A (B) titer testing results
With the increase of maternal serum IgG anti-A (B) titer, the
ratio of ABO haemolytic disease neonates gradually escalated.
Pearson correlation analysis showed that there was a positive
correlation between maternal serum IgG anti-A (B) titer and
neonatal ABO haemolytic incidence (r=0.883, P<0.01) (Table
2).
Maternal serum IgG anti-A (B) titer and severity of
the disease
The maternal serum titer of IgG anti-A (B) were not
significantly correlated with the severity of the disease, the
difference was not statistically significant (P>0.05, Table 3).
Table 2. Comparison of maternal serum IgG anti-A (B) titer and
neonatal ABO haemolytic incidence.
Titer
Pregnant women
ABO haemolytic disease newborns
Cases (n)
Ratio (%)
Cases (n)
Ratio (%)
1:16
0
0.00
0
0.0
1:32
83
15.2
1
1.2
1:64
97
17.8
4
4.1
1:128
115
21.1
33
28.7
1:256
91
16.7
47
51.7
1:512
74
13.6
63
85.1
1:1024
49
8.9
44
89.8
Biomed Res- India 2017 Volume 28 Issue 4
Effects of maternal serum IgG anti-A (B) and neonatal direct anti globulin red blood cell antibody expression on the
occurrence and development of neonatal ABO haemolysis
1:2048
37
6.8
35
94.6
Total
Total
546
100
227
41.6
Notes: Compared with pre-operation, *P<0.05; compared with control group,
#P<0.05
Table 3. Correlation of maternal serum titer of IgG anti-A (B) and
severity of the disease.
Phase
Cases
Mild
Moderate
Severe
<1:64
1
1 (100.0)
0
0
1:64
4
2 (50.0)
2 (50.0)
0
1:128
33
17 (51.5)
16 (48.5)
0
>1:128
189
95 (50.3)
94 (49.7)
0
227
115 (50.7)
112 (49.3)
0
Testing results of ABO haemolytic disease new-borns
Among all the 227 new-borns with ABO haemolytic disease,
the positive rate of RBC antibody testing was 100.0%, which is
higher than that of the free antibody testing 74.9%. The
difference was statistically significant (P<0.05) and the
positive rate of free antibody testing was also higher than that
of direct anti globulin testing 14.9%. The difference was
statistically significant (P<0.05, Table 4).
Table 4. Testing results of ABO haemolytic disease new-born’s.
Blood group (motherneonate)
Cases
Direct anti globulin
Free antibody
Red cell antibody
Positive (n)
Positive rate (%)
Positive (n)
Positive rate (%)
Positive (n)
Positive rate (%)
O-A
92
15
16.3*#
67
72.8
92
100
O-B
135
19
14.1*#
103
76.3#
135
100
Total
227
34
14.9*#
170
74.9#
227
100
Notes: Compare with free antibody, *P<0.05; Compared with red cell antibody,#P<0.05
Discussion
The ABO-HDN is an immune haemolytic disease of new-borns
caused by ABO blood group antibodies, especially among
blood group A or B new-borns born to blood group O mothers.
As one of the commonest haemolytic diseases of new-borns in
China, the process of ABO haemolysis is comparatively mild;
some severe neonates may have mild or moderate degrees of
haemolysis [8]. Severe cases like oedema, kernicterus or dead
foetus are not usual. However, early diagnose and treatment
can also prevent permanent neurological developmental
disorders or even death [9].
The main pathogenesis for ABO-HDN is the blood group
incompatibility of mother and foetus. Foetal red blood cells
enter maternal body through the placenta causing blood group
antigen stimulation and then inducing passive sensitization and
antigen clearance. After foetal red cells were coated by
maternal IgG blood group antibodies, a series of antigenantibody reaction gradually induced clinical symptoms [10].
Maternal serum IgG anti-A (B) titer was regarded as one of the
important indicators for early prediction of ABO haemolytic
disease of the new born and cited as one of prenatal diagnostic
items. Our research results showed that with the increase of
maternal serum IgG anti-A (B) titer, the ratio of ABO
haemolytic disease neonates gradually escalated, which was
also proved by the correlation analysis. This indicated that
early monitoring of serum IgG anti-A (B) titers has a positive
significance for the prediction of neonatal ABO haemolytic
disease. Nevertheless, it was pointed out by Arora et al. [11]
that red blood cell surface antigen, IgG antibody typing,
placenta density and other factors can also lead to changes in
Biomed Res- India 2017 Volume 28 Issue 4
titer, and it is complex and costs high to test IgG antibody
subtypes in clinic [11], the reference value is limited by simply
applying total IgG titer to predict ABO haemolytic disease of
the new born.
In order to further promote predictive diagnosis of ABO-HDN,
we conducted this research by selecting direct anti globulin,
free antibody and RBC antibody and the result demonstrated
that RBC antibody was the most sensitive to ABO haemolytic
diagnosis with a positive rate of 100.0%, the next one was free
antibody, the positive rate was 74.9% and the positive rate of
direct anti globulin was only 14.9% [12]. The first main reason
may be that most neonates have a low density of anti-A (B)
antigen and insufficient number of antibodies led to the weak
positive or negative testing result of direct anti globulin [13].
Secondly, free antibody has a positive significance for early
detection of neonatal ABO haemolysis because it can reflect
whether neonatal serum exist other antibodies or IgG anti A
(B) which do not match with red blood cells besides ABO [14].
Thirdly, RBC antibody testing is released by heating the
sensitized red blood cells, which can directly detect antibodies
who do not match with neonatal serum. So it is recommended
that we can apply the combination of maternal serum IgG antiA (B) titer and RBC antibody testing to clinical practice to
improve the efficiency of neonatal ABO haemolytic diagnosis.
Our study also showed that there was no evident correlation
between maternal serum IgG anti-A (B) titer and severity of
the disease. This was in accordance to the research of Tufekci
et al. [15]. Pregnancy IgG titer level cannot serve as the
standard for judging the prognosis of foetus [16]. In order to
make an early diagnosis of ABO-HDN, we should also pay
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attention to the regular inspection of blood routine, blood
group, specific antibody and blood coagulation mechanism on
the basis of IgG titer and RBC antibody testing.
In conclusion, it has some significance to test maternal serum
IgG anti-A (B), neonatal direct anti-globulin, free antibody and
RBC antibody for early diagnosis of neonatal ABO haemolytic
diagnosis. Maternal serum IgG anti-A (B) and RBC antibody
has high reference values. For pregnant women and new-borns
that have abnormalities in these two indicators, we should have
an early confirmed diagnosis and take actively preventive and
treatment measures to reduce the risk of neonatal ABO
haemolytic disease and improve population quality.
References
1. Li P, Pang LH, Liang HF, Chen HY, Fan XJ. Maternal IgG
anti-A and anti-B Titer levels screening in predicting abo
hemolytic disease of the newborn: A meta-analysis. Fetal
Pediatr Pathol 2015; 34: 341-350.
2. Jeon H, Calhoun B, Pothiawala M, Herschel M, Baron BW.
Significant ABO hemolytic disease of the newborn in a
group B infant with a group A2 mother.
Immunohematology 2000; 16: 105-108.
3. Beken S, Hirfanoglu I, Turkyilmaz C, Altuntas N, Unal S.
Intravenous immunoglobulin g treatment in abo hemolytic
disease of the newborn, is it myth or real? Indian J Hematol
Blood Transfus 2014; 30: 12-15.
4. Akgal S, Korkmaz A, Yiayit S, Yurdakak M. Neonatal
hyperbilirubinemia due to ABO incompatibility: does blood
group matter? Turk J Pediatr 2013; 55: 506-509.
5. Christensen RD, Yaish HM, Gallagher PG. A
paediatrician’s practical guide to diagnosing and treating
hereditary spherocytosis in neonates. Pediatrics 2015; 135:
1107-1114.
6. Chuansumrit A, Siripoonya P, Nathalang O, Sriphaisal T.
The benefit of the direct antiglobulin test using gel
technique in ABO hemolytic disease of the newborn.
Southeast Asian J Trop Med Public Health 1997; 28:
428-431.
7. Dinesh D. Review of positive direct anti-globulin tests
found on cord blood sampling. J Paediatr Child Health
2005; 41: 504-507.
8. Fu BM, Yuan XX, Tan YF. Serological testing and its
clinical application of ABO hemolytic disease of the
1858
newborn. J Maternal Child Health Care China 2013; 28:
2925-2927.
9. Brouwers HA, Overbeeke MA, van Ertbruggen I,
Schaasberg W, Alsbach GP. What is the best predictor of
the severity of ABO-haemolytic disease of the new-born?
Lancet 1988; 2: 641-644.
10. Ye CQ, Zhang H, Li WZ. The value of 3 trials about cord
blood hemolysis in the diagnosis of ABO hemolytic disease
of the newborn. J Chinese Pract Diag Ther 2013; 27: 45-46.
11. Taylor E, Vu D, Legare C. Intravenous immune globulinrelated hemolysis: comparing two different methods for
case assessment. J Transfusion 2015; 55: S23-S27.
12. Feng CS, Wan CP, Lau J, Lam TK, Fok TF. Incidence of
ABO haemolytic disease of the newborn in a group of
Hong Kong babies with severe neonatal jaundice. J Paediatr
Child Health 1990; 26: 155-157.
13. Jia JP, Huang XX, Li FF. The value of cord blood bilirubin
and 3 hemolytic inspections in the early diagnosis of ABO
hemolytic disease of the new-born. J Maternal Child Health
Care China 2014; 29: 227-230.
14. Ukita M, Takahashi A, Nunotani T, Kihana T, Watanabe S.
IgG subclasses of anti-A and anti-B antibodies bound to the
cord red cells in ABO incompatible pregnancies. Vox Sang
1989; 56: 181-186.
15. Tufekci S, Coban A, Bor M, Yasa B, Nisli K. Cardiac
rhythm abnormalities during intravenous immunoglobulin
G (IVIG) infusion in two new-born infants: coincidence or
association? Clin Case Rep 2015; 3: 731-734.
16. Bu XM, Liang HG, Song JL. The diagnostic value of the
detection of erythrocyte membrane antibody levels by
flowcytometry method in ABO hemolytic disease of the
new-born. J China Pediatric Blood Cancer 2013; 18:
281-283.
*Correspondence
to
Ming Chen
Department of Blood Transfusion
The People's Hospital of Ruian
China
Biomed Res- India 2017 Volume 28 Issue 4